Immunoenzymetric assay for the in vitro quantitative measurement of human interferon gamma (IFN-_) in serum and plasma.
The IFN-_-ELISA is a solid phase Enzyme Amplified Sensitivity Immunoassay performed on microtiterplate. The assay uses monoclonal antibodies (MAbs) directed against distinct epitopes of IFN-_. Standards and samples react with the capture monoclonal antibody (MAb 1) coated on microtiter well and with a monoclonal antibody (MAb 2) labelled with horseradish peroxidase (HRP). After an incubation period allowing the formation of a sandwich: coated MAb 1 – human IFN-_ – MAb 2 – HRP, the microtiterplate is washed to remove unbound enzyme labelled antibody. Bound enzyme-labelled antibody is measured through a chromogenic reaction.Chromogenic solution (TMB) is added and incubated. The reaction is stopped with the addition of Stop Solution and the microtiterplate is then read at the appropriate wavelength.The amount of substrate turnover is determined calorimetrically by measuring the absorbance, which is proportional to the IFN-_ concentration.A calibration curve is plotted and IFN-_ concentration in samples is determined by interpolation from the calibration curve. The use of the ELISA reader (linearity up to 3 OD units) and a sophisticated data reduction method (polychromatic
data reduction) result in a high sensitivity in the low range and in an extended calibration range.